Peyer's patches (PP) and isolated lymphoid follicles (ILF) are organized mucosal lymphoid tissues in the intestine that serve as inductive sites for immune responses to antigens in the intestinal lumen. We discovered that RANKL (also known as TRANCE and TNFSF11), a member of the TNF superfamily previously shown to have obligatory roles in the development of lymph nodes, the differentiation of medullary thymic epithelial cells, the differentiation of osteoclasts, and mammary gland lactation, is also present on stromal cells in ILF and PP. These RANKL-expressing stromal cells are concentrated in the subepithelial dome area in close apposition to the follicle-associated epithelium. PP in RANKL null mice were previously characterized as smaller than those in wild type mice, but additional phenotypic abnormalities were not reported. We find that the PP of RANKL null mice have a large deficit in the development of M cells, specialized epithelial cells capable of rapid uptake and delivery of particulate antigens into antigen-presenting cells (APC) found within and beneath the epithelium. On average, the small intestine of RANKL null mice has 74-fold fewer M cells detected by staining with the UEA-1 lectin than wild type control mice. The loss of UEA-1+ M cells in RANKL null mice correlates with a nearly complete loss in the ability of RANKL null PP to translocate 200 nm diameter fluorescent beads from the intestinal lumen into APC located in the PP subepithelial dome. M cell development in RANKL null mice can be rescued by treatment with recombinant RANKL for 7 days. Treatment of wild type mice with neutralizing anti- RANKL antibodies reproduces the loss of M cells observed in RANKL null mice. The central hypothesis guiding the proposed experiments is that RANKL acting through its receptor RANK on epithelial cells is a critical signaling pathway required for normal M cell differentiation and function. While M cells were first described in the rabbit appendix over 30 years ago and are known to have a central role in uptake of particulate antigens in mucosal tissues while also being exploited as a portal of entry by pathogens, the details of the histogenesis of these cells have largely remained a mystery. The first aim of the proposal is to determine whether stromal cells are the major source of RANKL involved in induction of M cell differentiation in the FAE and whether the action of RANKL to induce M cell differentiation is restricted to stem cells in the dome-associated crypts. The second aim is to determine whether the functional activity of M cells can be altered by manipulation of RANKL- RANK signaling with exogenous RANKL and neutralizing anti-RANKL antibody. The third aim is to determine how local expression of the osteoprotegerin (OPG) soluble decoy receptor and RANKL-induced effects on intestinal DC and macrophages influence the effects of stromal cell RANKL on RANK-expressing enterocytes. Mechanistic insights into how the RANKL-RANK pathway supports pathways of antigen handling by the gut immune system that foster development of tolerance of antigens normally encountered in the gut lumen may be useful in developing oral vaccination strategies and in the treatment of human inflammatory bowel disease. PUBLIC HEALTH RELEVANCE: The goal of this research is to identify the role of a protein known as RANKL (RANK ligand) and RANK (its receptor) in the establishment and normal functioning of organized lymphocyte containing structures in the small and large intestine. These lymphoid structures such as Peyer's patches normally contribute to protecting the host from pathogens and preventing inappropriate immune responses to antigens normally encountered in the intestinal microenvironment. These studies are expected to contribute to existing knowledge of how specific imbalances in the intestinal immune system can lead to various forms of human inflammatory bowel disease.